Phospholipids are a diverse class of amphipathic molecules present in all biologicial membranes. Because of the experimental difficulties encountered in working with membrane components, relatively little is known about the enzymatic and genetic control of phospholipid metabolism or about the role of individual phospholipid species in membrane function. A greater understanding of this fundamental aspect of membrane biochemistry would be desirable, since phospholipids play an important role in many disease processes, including hyperlipoproteinemias, diabetes, and certain inborn errors of metabolism. The isolation and characterization of animal cell mutants lacking specific phospholipid molecules are the major goals of this project. The physiology of such mutants is expected to shed light on phospholipid regulation and function. In the long run these mutants should facilitate the isolation of the genes that code for the enzymes involved in the synthesis and assembly of higher eucaryotic membrane lipids. It is estimated that there must be several hundred of such genes in an animal cell, but only a few mutants have been isolated so far. In the coming grant period, three major projects will be emphasized. 1) The laboratory will develop schemes for the isolation of animal cell mutants deficient in the enzymes of phosphatidylinositol synthesis, phosphorylation, and turnover. 2) The biochemistry and genetics of animal cell mutants deficient in the biosynthesis of plasmalogens will be studied. During the last grant period a collection of Chinese hamster ovary cell mutants deficient in the peroxisomal dihydroxyacetone phosphate acyltransferase was isolated, an enzyme that is also missing in Zellweger's syndrome. 3) The interaction of Escherichia coli lipid A molecules with membranes of animal cells will be characterized. Recent studies from the PI's laboratory have led to the elucidation of the biosynthesis of lipid A in E. coli. The resulting new enzymology provides a unique opportunity to make labeled probes for studying the mechanisms by which lipid A molecules trigger responses in animal cells, such as macrophage activation or mitogenesis. The above studies will advance fundamental understanding of the role of lipid molecules in cell membranes.